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A.I.S.F. Monothematic Conference 2012The Gut-Liver Axis:
a bidirectional relation in health and disease
Alcoholic steatohepatitis
Gianluca Svegliati Baroni
SOS “Epatopatie croniche e trapianto di fegato”
Universita Politecnica Marche-Ospedali Riuniti
Ancona
Bologna, 5 ottobre 2012
Il sottoscritto dichiara di non aver avuto negli ultimi 12 mesi conflitto d’interesse in relazione a questa presentazione
e
che la presentazione non contiene discussionedi farmaci in studio o ad uso off-label
LE CAUSE DELLE MALATTIE EPATICHE:
ALCOL
Consumo di bevande alcoliche
distinzione per classi di età
Abuso di alcol, come causa di patologie al fegato
che portano al decesso del paziente…l’ alcol ha un’ azione epatotossica diretta….agiscecome cofattore di danno accelerando la progressionedelle malattie epatiche croniche da altra causa…ilconsumo di alcol è la più importante determinantedella severità della malattia epatica, causando unincremento di mortalità di tre volte superiore nei forticonsumatori rispetto ai bassi consumatori.
Total number of first liver transplants = 1530
* Autoimmune 0.35%; liver rupture 0.15%
Re-OLTx N. = 100 (7.5%)
THE LIVER MATCH STUDYMain indications for liver transplantation
Angelico M. et al. Dig Liv Dis; 2011.
0
1,3
6,6
9,3
1,6
6,9 7,2 7,1
1,6
4,3
2,63,1
0
5
10
< 30 30 - 49 50 - 69 > 70
HCV/HBV
Alcol
NAFLD
Prevalenza età-specifica di alterazione degli indici di danno epatico nella popolazione generale di Cittanova (Ita lia
Meridionale) per eziologia, 2002-2003
Pendino et al. Hepatology 2005
%
Population Screened 4671
Excessive drinkers (>30g/day) 947 20.3 %
EtOH-Induced Liver Damage 305 6.5 %
Cirrhosis+HCC (10% symptom.) 33 0.7 %
Bellentani et al., Gut, 1997 and Add.Biol, 2002
“True” Clinical Prevalence of Ethanol induced Liver Diseases
Dionysos 1 (1991-92)
Alcohol Intake O.R. for ALD O.R. for Cirrhosis
Teetottalers 0 0<30 g/day 0 031-60 g/day 8 1061-90 g/day 21 2591-120 g/day 15 53>120 g/day 36 62
Which is the threshold risk of alcohol consumption for ALD in the general Italian healthy population?
Bellentani et al., Gut, 1997
More than 30 gEtOH/day for more that 10 years
Genetic? Environmental?
From Glass To Bed
(~20%)
(~50%) 10-15 yrs
More than 40-80 gEtOH/day for more that 25 years
(~40-60%)
Chronic Alcoholic Hepatitis
Alcoholic Cirrhosis (3%)
Steatosis (90%)
AASLD Guidelines, Hepatology 2010
M. Rojkind, N.Y., USA C. S. Lieber, N.Y., USAA.M. Jezequel, Ancona, Italy
G. Svegliati Baroni 1994G. Svegliati Baroni 1997, 1999G. Svegliati Baroni 2001, 2005
PATOGENESI DELLA STEATOSI EPATICA:THE “MULTI-HIT“ THEORY
FIRST HIT SECOND HIT
?
Alterazioni potenziale redox
•Aumentato up-take e sintesi degli ac. grassi•Ridotta escrezione degli ac.grassi•Ridotta beta-ossidazione
Produzione di ROSDeplezione antiossidanti
•Lipoperossidazione •Induzione di citochine•Infiammazione•Apoptosi
THIRD HIT
•Cirrosi
?
Similarities and Differences in the Pathogenesis of Alc oholicand Nonalcoholic Steatohepatitis (Diehl et al., Sem Liv Dis 2009)
The natural history of ASH appears to be much more ‘‘aggressi ve’’ thanthat of NASH suggesting that ethanol and/or acetaldehyde ma y haveunique roles in steatohepatitis progression.
Acetaldehyde interacts with various molecules to form adducts that haveimmunogenic properties, and it has also been shown to direct ly activatecollagen gene expression in hepatic stellate cells (Svegliati-Baroni G et al.,1993, 1993, 1994, 1997, 2001, 2005, in press).
Ethanol itself disorders lipid membranes and its (nonoxida tive)metabolism also generates fatty acid ethyl esters that may be cytotoxic(Benedetti A et al., 1993, 1995).
Therefore, chronic exposure to ethanol and/or its metaboli tes may furtherchallenge livers that are concomitantly experiencing stre sses related tolipotoxicity, oxidative and ER stress, and exposure to cyto kines andPAMP recognition receptors, exacerbating hepatocyte inju ry and relatedinflammation and fibrogenesis.
Similarities and Differences in the Pathogenesis of Alc oholicand Nonalcoholic Steatohepatitis (Diehl et al., Sem Liv Dis 2009)
Prevalenza della malnutrizione in pazienti alcolisti
Mendenhall 1984
Mettere diapo colline marchigiane + salame + vino
16%
46%
76%
95%
0%
20%
40%
60%
80%
100%
Steatosis at US
C (n=67)
HD (n=69)
O (n=66)
O+HD (n=55)
Overall Prevalence
58.3%
Fatty Liver in the Dionysos study
(Too much food is worst than too much alcohol !)
Bellentani et al., Ann.Int.Med., 2000
EXPERIMENTALS MODELS OF ALCOHOLIC LIVER INJURY
Lieber-DeCarli liquid diet Intragastric infusion model
Synergistic steatohepatitis by moderate obesity and alc oholin mice despite increased adiponectin and p-AMPK
Xu et al., J Hepatol 2011
TLR4 signalling
TR
IF
Proinflammatory cytokinesTNF-αααα , IL-6, IL-12, CXCL1
IKKεεεε
IRF3
IFN-ββββ transcriptionRANTES, CXCL10
JNK
TRAF6
Nucleus
MD2CD14
TLR4
LPS(HMGB1, Hyaluronan)
TIR
AP
TR
AM
IKK
αα αα
IKK
ββ ββIκκκκBαααα
p50 p65
TBK1
NF κκκκB
TAK1
IRA
K4
IRA
K1
MyD
88
AP-1IRF3
MyD88-dependent pathwayMyD88-independent pathway
Seki, De Minicis, Nat Med. 2007
LPS
Antibiotics Prevent Liver Injury in Rats Following Long -term Exposure toEthanol
Adachi et al., Gastroenterology 1995
Enteric Dysbiosis Associated with a Mouse Model ofAlcoholic Liver Disease
Yan et al., Hepatology 2011
Enteric Dysbiosis Associated with a Mouse Model ofAlcoholic Liver Disease
Yan et al., Hepatology 2011
Enteric Dysbiosis Associated with a Mouse Model ofAlcoholic Liver Disease
Yan et al., Hepatology 2011
Endotoxemia and Gut Barrier Dysfunction in Alcoholic Liver
Disease
Rao R. Hepatology 2009
Plasma endotoxin concentration in plasma in patients w ithalcoholic and non-alcoholic liver disease
0
5
10
15
20
25
Controls Fatty liver Alcoholichepatitis
Alcoholiccirrhosis
Non-alcoholiccirrhosis
Fukui et al., J Hepatol 1991
** p < 0.05 vs nonalcoholic cirrhosis
Increased intestinal permeability to macromolecules and endotoxemia in patients
with chronic alcohol abuse in different stages of alcohol-induced liver disease
0
5
10
15
20
25
30
Controls Steatosis Alcoholic
hepatitis
Cirrhosis
Endotoxin (pg/ml)
0
1
2
3
Controls Steatosis Alcoholic
hepatitis
Cirrhosis
% Urine PEG 400 recovery
Perlasek et al., J Hepatol 2000; Urbaschek et al., Alcoholism Clin Exp Res 2001
p < 0.05 vs controls
Endotoxin levels in patients with alcoholic liver cirrhos is and severe portal hypertension without an acute complicatio n
Trebicka et al., Eur JGastroenterol Hepatol 2011
TLR4 signalling
TR
IF
Proinflammatory cytokinesTNF-αααα , IL-6, IL-12, CXCL1
IKKεεεε
IRF3
IFN-ββββ transcriptionRANTES, CXCL10
JNK
TRAF6
Nucleus
MD2CD14
TLR4
LPS(HMGB1, Hyaluronan)
TIR
AP
TR
AM
IKK
αα αα
IKK
ββ ββIκκκκBαααα
p50 p65
TBK1
NF κκκκB
TAK1
IRA
K4
IRA
K1
MyD
88
AP-1IRF3
MyD88-dependent pathwayMyD88-independent pathway
Seki, De Minicis, Nat Med. 2007
HFD
ET-OH
Wild-type
Toll-Like Receptor 4 Is Involved in the Mechanism of Early Alcohol-Induced Liver Injuryin Mice
TLR4-/-
Uesugi, Hepatology 2001
Toll-Like Receptor 4 Is Involved in the Mechanism of Early Alcohol-Induced Liver Injury in Mice
Uesugi, Hepatology 2001
Toll-like receptor 4 mediates alcohol-induced steatohepa titis through bonemarrow-derived and endogenous liver cells in mice
Inokuchi, Alcoholism Clin Exp Res 2011
TLR2: microbial lipopeptides;
TLR4: lipopolysaccharides (LPSs) from Gram-
TLR1/6: triacyl- and diacyllipopeptides;
TLR3: viral double-stranded RNA;
TLR5: bacterial flagellin.
TLR7 and TLR8: viral single-stranded RNA
Differential Liver Sensitization to Toll-like Receptor Pat hways in Mice With AlcoholicFatty Liver
Gustot et al., Hepatology 2006
1) The administration of broad-spectrumantibiotics reduced the severity of alcohol-induced fatty liver but did not modify alcohol-induced TLR gene expression;
2) ROS from NADPH oxidase increased the severity of alcohol-induced fatty liver and the expression of liver TLRs;
3) Severe steatosis is the visible part of a “first hit” on the liver, combined with anupregulation of TLRs;
4) The administration of microbial products toEtOH-exposed animals may induce the “second hit” on the liver with inflammatoryinfiltrate and satellitosis formation.
Differential Liver Sensitization to Toll-like Receptor Pat hways in Mice With AlcoholicFatty Liver
Gustot et al., Hepatology 2006
TLR4 signalling
TR
IF
Proinflammatory cytokinesTNF-αααα , IL-6, IL-12, CXCL1
IKKεεεε
IRF3
IFN-ββββ transcriptionRANTES, CXCL10
JNK
TRAF6
Nucleus
MD2CD14
TLR4
LPS(HMGB1, Hyaluronan)
TIR
AP
TR
AM
IKK
αα αα
IKK
ββ ββIκκκκBαααα
p50 p65
TBK1
NF κκκκB
TAK1
IRA
K4
IRA
K1
MyD
88
AP-1IRF3
MyD88-dependent pathwayMyD88-independent pathway
Seki, De Minicis, Nat Med. 2007
Essential role of tumor necrosis factor alpha in alcohol-induced liver injury in mice.
0
1
2
3
4
5
6
7
wild-type p75-/- p55-/-
Deg
ree
ofliv
erin
jury
Yin et al., Gastroenterology1999
TNF-α
Kishoreet al., AJP 2002; Shi et al., AJP 2002 McMullen et al., Gastroenterology 2005
The Critical Role of Toll-Like Receptor (TLR) 4 in Alcoho lic Liver DiseaseIs Independent of the Common TLR Adapter MyD88
TR
IF
Proinflammatory cytokinesTNF-αααα , IL-6, IL-12, CXCL1
IKKεεεε
IRF3
IFN-ββββ transcriptionRANTES, CXCL10
JNK
TRAF6
Nucleus
MD2CD14
TLR4
LPS(HMGB1, Hyaluronan)
TIR
AP
TR
AM
IKK
αα αα
IKK
ββ ββIκκκκBαααα
p50 p65
TBK1
NF κκκκB
TAK1
IRA
K4
IRA
K1
MyD
88
AP-1IRF3
MyD88-dependent pathwayMyD88-independent pathway
Hritz et al., Hepatology 2008
Ethanol Augments RANTES/CCL5 Expression in Rat Liver Sin usoidal EndothelialCells and Human Endothelial Cells via Activation of NF-B, HIF-1, and AP-1
Yeligar et al., J Immunol 2008
Tilg & Moschen, Hepatology 2010
The Multiple Parallel Hits Hypothesis
Acetaldehyde
Samuel & Shulman, Cell 2012
Synergistic steatohepatitis by moderate obesity and alc oholin mice despite increased adiponectin and p-AMPK
Xu et al., J Hepatol 2011
ER stress markers
Cell deathsignalingmolecules
Epigenetic Regulation of Hepatic Endoplasmic Reticulum St ress Pathways in the Ethanol-Fed Cystathionine Beta Synthase–Deficient Mous e
Esfandiari et al., AJP 2005; Hepatology 2010
Toll-like receptor 4 mediates synergism between alcohol and HCV in hepatic oncogenesis involving stem cell marker Nanog
Machida et al., PNAS 2009
Toll-like receptor 4 mediates synergism between alcohol and HCV in hepatic oncogenesis involving stem cell marker Nanog
Machida et al., PNAS 2009
A Randomized, Double-Blinded, Placebo-Controlled Mul ti-CenterTrial of Etanercept in the Treatment of Alcoholic Hepat itis
0
10
20
30
40
50
60
Etanercept Placebo
6-months mortality (%)
0
5
10
15
20
25
30
35
40
Etanercept Placebo
Infectious events (%)
Boetticher et al., Gastroenterology2008
Conclusions
• Alcohol consumption leads to increased intestinalpermeability and endotoxemia, which are involved inthe pathogenesis of ALD ;
• Intestinal microflora not only is the source ofcirculating endotoxins but also plays a role in thegeneration and accumulation of acetaldehyde in thecolonic lumen and has a subsequent influence onepithelial barrier dysfunction;
• Gut-liver axis stimuli represents a major second-hitin the pathogenesis of ASH ;
• It is therefore important to delineate the mechanismsinvolved in endotoxin-mediated liver cell injury todesign preventive and therapeutic strategies for thetreatment of ALD .
Ettore Svegliati BaroniCurriculum vitae
Nato: Ancona, 02/05/2012
Comitato Coordinatore AISF2052-2055 ?
Severity of liver injury in experimental alcoholic liver di sease.Correlation with plasma endotoxin, prostaglandin E2,leukotriene B4, and thromboxane B2.
Nanji et al, Am J Pathol 1993
Plasma levels of soluble tumor necrosis factor receptors p 55 and p75 in patients with alcoholic liver disease of increasin g severity
0
1000
2000
3000
4000
5000
6000
7000
Controls Steatosis Fibrosis Cirrhosis Cirrhosis with
AAH
Cirrhosis with
AAH +++
TNFsRp75 (pg/ml)
*
*
Naveau et al., J Hepatol 1998
* *
Synergistic steatohepatitis by moderate obesity and alc oholin mice despite increased adiponectin and p-AMPK
Xu et al., J Hepatol 2011
M1macrophages
M2macrophages
Overall Prevalence DionysosCohort
Liver Disease Prevalence 1992 (%)
Prevalence 2002 (%)
Persistent alteration of liver enzymes (LE)
27.6 27.2
Persistent alteration of LE with fatty liver (FL)
45 42
NAFLD (alcohol intake < 30 g/die)
25 32
AFLD (alcohol intake > 30 g/die)
20 10
Cirrhosis 1.1 1.0
L’acetaldeide stimola l’espressione del gene per il collagene (COL1A2).
1 3 12 24
1
23
Fol
d in
crea
se
α2(α2(α2(α2(I) ) ) ) collagen mRNA48
4+ Ac
+ Ac+ cyhx
(Time, Hrs)
B
A
ACCONT
TRANSCRIPTION RATEFOLD INCREASE
1 3 5
C
5
G. Svegliati Baroni 1994G. Svegliati Baroni 1997G. Svegliati Baroni 2001, 2005
1
αα αα2(
I) C
OLL
AG
EN
mR
NA
FO
LD IN
CR
EA
SE
2
3
CONT + AC
+ CTase
4 AC - + - +
+ CTase
αααα2(I)
S14
A B
G. Svegliati Baroni 1994G. Svegliati Baroni 1997G. Svegliati Baroni 2001, 2005
G. Svegliati Baroni 1994G. Svegliati Baroni 1997G. Svegliati Baroni 2001, 2005
EFFETTO DI SPECIFICI INIBITORI SULLA SINTESI DI COLLAGENE E FIBRONECTINA
A.M. Jezequel, Ancona, Italy
Plasma levels of soluble tumor necrosis factor receptors p 55 and p75 in patients with alcoholic liver disease of increasin g severity
0
500
1000
1500
2000
2500
3000
3500
4000
4500
Controls Steatosis Fibrosis Cirrhosis Cirrhosis with
AAH
Cirrhosis with
AAH +++
TNFsRp55 (pg/ml)
*
*
Naveau et al., J Hepatol 1998
**
alcohol-induced liver injury is significantly
ameliorated in mice mutated in the TLR4 gene (Hritz et al., 2008; Uesugi et al., 2001).
Downstream of TLR4, adaptor proteins MyD88 and TRIF are activated for transmitting
their
signals. The TRIF-dependent pathway is more important than the MyD88-dependent
pathway in alcohol-induced liver injury after the Lieber-DeCarli diet feeding (Hritz et al.,
2008; Zhao et al., 2008). The TRIF-dependent pathway induces IRF3 activation and TNF-α
production after chronic ethanol treatment (Zhao et al., 2008).
Questo Zhao 2008 può essere importante per talk Milano-Missale
As reported previously, both TLR4 and
Kupffer cells are critical for the development of experimental alcohol-induced
steatohepatitis (Hritz et al., 2008; Uesugi et al., 2001) (Adachi et al., 1994; Koop et al.,
1997)
ETANOLO ACETALDEIDE ACETATO
NADPH NADP+
ADHBassa Km
NAD+ NADH
ALDHBassa Km
CYP2E1Alta Km
O2-
NAD+ NADH
Aldeide ossidasiXantina ossidasi
Alta Km
O2-
METABOLISMO DELL’ ETANOLO
Pathogenesis of alcohol-induced liver disease: classica lconcepts and recent advances (Seth et al., JGH 2011)